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Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants

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Abstract

Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.

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Acknowledgements

We express our gratitude to the Monash University Malaysia (Monash Seed Grant under the Bioactive Compounds Research Strength), the Ministry of Science, Technology & Innovation (grant no. 02-02-10-SF0077), and the Ministry of Higher Education (grant no. FRGS/1/2014/SKK03/MUSM/02/1) for funding and supporting this project.

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Authors and Affiliations

  1. School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500, Bandar Sunway, Selangor, Malaysia

    Amelia Nathania Dong, Uma Devi Palanisamy & Nafees Ahemad

  2. Department of Biomedical Science, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia

    Yan Pan

  3. School of Pharmacy, International Medical University, 126, Jalan Jalil Perkasa 19, 57000, Bukit Jalil, Kuala Lumpur, Malaysia

    Beow Chin Yiap & Chin Eng Ong

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  1. Amelia Nathania Dong
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Correspondence to Chin Eng Ong.

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Fig. 1S

Representative reduced CO difference spectra showing expression of the (a) CYP2D6 wild type (CYP2D6*1) together with its alleles, and (b) CYP2C19 wild type (CYP2C19*1) with its alleles in E. coli membranes. (GIF 4009 kb)

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Dong, A.N., Pan, Y., Palanisamy, U.D. et al. Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants. Appl Biochem Biotechnol 186, 132–144 (2018). https://doi.org/10.1007/s12010-018-2728-0

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